Suppressing Proteasome Mediated Processing of Topoisomerase II DNA-Protein Adducts Preserves Genome Integrity
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ABSTRACT:
PROVIDER: PRJNA589537 | ENA |
REPOSITORIES: ENA
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Project description:Here, we utilize high-resolution mapping of ETO-induced TOP2 lesions genome-wide (END-seq) (Canela et al., 2016), to examine the impact of proteasome inhibition on the fate of TOP2ccs. We found that once ETO-stabilized TOP2ccs had been proteolytically processed throughout the genome, and a robust DDR had been initiated, the large number of newly generated protein-free DSBs were repaired in a highly error-prone manner, resulting in toxic chromosomal translocations and rearrangements that lead to cell death. Notably, mis-repair of ETO-induced lesions occurred independently of the classical NHEJ or HR pathways. By inhibiting proteasome-mediated degradation of TOP2ccs either through chemical inhibition or by ablation of the ubiquitination of TOP2ccs by RNF4, an intact and enzymatically competent TOP2 was able to re-seal the protein-linked DSBs without invoking a significantly DDR. As a result, cells were protected from genomic instability, which ultimately led to enhanced cell survival after treatment with topoisomerase poisons.
2020-02-20 | GSE140372 | GEO
Project description:Topoisomerase II (TOP2) relieves topological stress in DNA by introducing double-strand breaks (DSBs) via a transient, covalently linked TOP2 DNA-protein intermediate, termed TOP2 cleavage complex (TOP2cc). TOP2ccs are normally rapidly reversible, but can be stabilized by TOP2 poisons, such as the chemotherapeutic agent etoposide (ETO). TOP2 poisons have shown significant variability in their therapeutic effectiveness across different cancers for reasons that remain to be determined. One potential explanation for the differential cellular response to these drugs is in the manner by which cells process TOP2ccs. Cells are thought to remove TOP2ccs primarily by proteolytic degradation followed by DNA DSB repair. Here, we show that proteasome-mediated repair of TOP2cc is highly error-prone. Pre-treating primary splenic mouse B-cells with proteasome inhibitors prevented the proteolytic processing of trapped TOP2ccs, suppressed the DNA damage response (DDR) and completely protected cells from ETO-induced genome instability, thereby preserving cellular viability. When degradation of TOP2cc was suppressed, the TOP2 enzyme uncoupled itself from the DNA following ETO washout, in an error-free manner. This suggests a potential mechanism of developing resistance to topoisomerase poisons by ensuring rapid TOP2cc reversal.
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Project description:Cisplatin, one of the most widely used anticancer drugs, crosslinks DNA and ultimately induces cell death. However, the genomic pattern of cisplatin-DNA adducts remains unknown, due to the lack of a reliable and sensitive genome-wide method. Here we present “cisplatin-seq” to identify genome-wide cisplatin crosslinking sites at base-resolution. Cisplatin-seq reveals that mitochondrial DNA is a preferred target of cisplatin. For nuclear genome, cisplatin-DNA adducts are enriched within promoters and regions harboring transcription termination sites. While the density of GG dinucleotide determines the initial crosslinking of cisplatin, binding of proteins to the genome largely contributes to the accumulative pattern of cisplatin-DNA adducts.
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Project description:The proteasome maintains protein quality during aging and disease. Challenges to proteasome function can be compensated by local proteasome stress responses. However, whereas proteasome stress is also sensed systemically is unknown. In Drosophila , we find that proteasome stress in skeletal muscle non-autonomously promotes the degradation of proteasome substrates in distant tissues during aging. Several muscle-secreted factors (myokines) are upregulated by proteasomal stress via C/EBP transcription factors, including the amylase Amyrel, which increases the circulating levels of the disaccharide maltose. Muscle-specific Amyrel overexpression promotes the degradation of proteasome substrates in the aging brain and retina via the transcriptional induction of chaperones and proteases. Conversely, RNAi for maltose transporters worsens proteostasis and reduces the expression of Amyrel-induced genes in the brain. Moreover, maltose preserves protein quality in cell culture and human cortical brain organoids challenged by thermal stress. Thus, proteasome stress in skeletal muscle mounts a systemic adaptive response via amylase/maltose signaling.
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Project description:The proteasome maintains protein quality during aging and disease. Challenges to proteasome function can be compensated by local proteasome stress responses. However, whereas proteasome stress is also sensed systemically is unknown. In Drosophila , we find that proteasome stress in skeletal muscle non-autonomously promotes the degradation of proteasome substrates in distant tissues during aging. Several muscle-secreted factors (myokines) are upregulated by proteasomal stress via C/EBP transcription factors, including the amylase Amyrel, which increases the circulating levels of the disaccharide maltose. Muscle-specific Amyrel overexpression promotes the degradation of proteasome substrates in the aging brain and retina via the transcriptional induction of chaperones and proteases. Conversely, RNAi for maltose transporters worsens proteostasis and reduces the expression of Amyrel-induced genes in the brain. Moreover, maltose preserves protein quality in cell culture and human cortical brain organoids challenged by thermal stress. Thus, proteasome stress in skeletal muscle mounts a systemic adaptive response via amylase/maltose signaling.
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2024-01-19 | GSE251645 | GEO
Project description:Eukaryotic topoisomerase 1 (TOP1) regulates DNA topology to ensure efficient DNA replication and transcription. TOP1 is also a major driver of endogenous genome instability, particularly when its catalytic intermediate - a covalent TOP1-DNA adduct known as a TOP1 cleavage complex (TOP1cc) - is stabilised. TOP1ccs are highly cytotoxic and a failure to resolve them underlies the pathology of neurological disorders but is also exploited in cancer therapy where TOP1ccs are the target of widely used frontline anti-cancer drugs. A critical enzyme for TOP1cc resolution is the tyrosyl-DNA phosphodiesterase, TDP1, which hydrolyses the bond that links a tyrosine in the active site of TOP1 to a 3’ phosphate group on a single-stranded (ss)DNA break. However, TDP1 can only process small peptide fragments from ssDNA ends, raising the question of how the ~90 kDa TOP1 protein is processed upstream of TDP1. We find that TEX264 fulfils this role by forming a complex with the p97 ATPase and the SPRTN metalloprotease. We show that TEX264 recognises both unmodified and SUMO1-modifed TOP1 and initiates TOP1cc repair by recruiting p97 and SPRTN. TEX264 localises to the nuclear periphery, associates with DNA replication forks, and counteracts TOP1ccs during DNA replication. Altogether, our study elucidates the existence of a specialised repair complex required for upstream proteolysis of TOP1ccs and their subsequent resolution.
2020-03-13 | PXD017239 | Pride
Project description:MR1T cells are a recently discovered class of T cells that recognize antigens presented by the MHC-I–related molecule MR1 in the absence of microbial infection. The nature of the self-antigens that stimulate MR1T cells remains unclear, hampering our understanding of their physiological role and therapeutic potential. By combining genetic, pharmacological, and biochemical approaches, we found that carbonyl stress and changes in nucleobase metabolism in target cells promote MR1T cell activation. Stimulatory compounds formed by carbonyl adducts of nucleobases were detected within MR1 molecules produced by tumor cells and their abundance and antigenicity were enhanced by drugs that induce carbonyl accumulation. Our data reveal carbonyl nucleobase adducts as MR1T cell antigens. Recognizing cells under carbonyl stress allows MR1T cells to monitor cellular metabolic changes with physiological and therapeutic implications.
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2022-06-24 | GSE206519 | GEO
Project description:The cancer target enyzme topoisomerase 1 transiently cleaves one strand of chromosomal DNA to relax accumulated strain that can prevent transcription, replication or chromatin assembly. The topoisomerase 1 poison camptothecin and its synthetic analogs are widely used chemotherapeutics that act by trapping the enzyme on DNA in a ‘covalent complex’, resulting in persistent DNA damage and cell death. The prevailing model, interfacial inhibition, contends that the covalent complex is trapped by drug binding alone. In contrast, here we show that camptothecins produce extensive oxidative stress and that topoisomerase 1 is reactive with electrophilic second messengers of oxidative damage. We show that blocking oxidative stress in cells inhibits covalent complex formation by camptothecin, while electrophiles such as 4-hydroxynonenal are able to induce covalent complex formation in cells on their own. Towards mechanism of action, we show that 4-hydroxynonenal electrophilically modifies a cysteine within the DNA-binding active site of human topoisomerase 1. Taken together, our results suggest a mechanism in which oxidative stress mediates the effects of many topoisomerase 1 poisons, and suggest that this critical DNA-regulating enzyme may have a dual role as a sensor of cellular redox stress, linking oxidative stress to DNA damage response in cancer cells.
2021-03-24 | PXD018549 | Pride